Although &#945;-synuclein plays a clear role in both the genetics and pathology of Parkinson disease, the mechanism by which &#945;-synuclein causes neuronal cell death remains unknown. We have examined the effects of &#945;-synuclein on the biology of cultured neuroblastoma cells and primary neurons. Overexpression of disease-causing forms of &#945;-synuclein, including wild-type, can drive apoptosis and necrosis in cultured cells. These effects can be exaggerated by increasing the amounts of dopamine derived radicals in cells, suggesting that increased amounts of a-synuclein, especially mutant forms, sensitizes cells to cell death. Overexpression of wild-type &#945;-synuclein also causes an increased susceptibility to mitochondrial complex I inhibition. We have begun analysis of a possible direct effect of &#945;-synuclein on mitochondrial function by using live cell imaging of mitochondrial network integrity and are currently examining if mitochondrial complex I inhibition leads to more severe deficits in cells that overexpress &#945;-synuclein. We will also use primary neurons from &#945;-synuclein null mice as a model for testing the effect of &#945;-synuclein variants on mitochondrial connectivity. A major advantage of this model is that it provides a more physiologic model that can be useful in extrapolating to the role of &#945;-synuclein in vivo.[unreadable] Aberrant post-translational modification of &#945;-synuclein may also be a factor in the pathogenic mechanism of &#945;-synuclein in Parkinsons disease. Phosphorylated &#945;-synuclein (Ser129) is a major component of Lewy bodies and glial cytoplasmic inclusions and is therefore thought to play a critical pathogenic role in all synucleinopathies. In cases with a triplication of the a-synuclein gene, we have found that phosphorylated &#945;-synuclein (Pser129-asyn) is disproportionately elevated in Lewy body containing brain regions suggesting this form of &#945;-synuclein is at the very least a reliable marker of &#945;-synuclein pathology. In cultured cells we have found that the detection of Pser129-asyn is directly correlated with the amount of total &#945;-synuclein suggesting that overexpression of &#945;-synuclein drives its phosphorylation. In future studies we will use primary neurons from SNCA null mice to examine how lack of Ser129 phosphorylation affects the turnover and possible aggregation of &#945;-synuclein. In conjuction with mitochondrial network experiments mentioned above, we will also examine how phosphorylation at Ser129 affects &#945;-synucleins cellular localization, one possibility being a phosphodependent interaction with mitochondria.